Fluorescent TrueBlot®: Anti-Mouse Ig Fluorescein

Description

Background

Mouse IgG TrueBlot® is a unique fluorescein conjugated Anti-mouse IgG monoclonal secondary antibody. Mouse IgG TrueBlot® enables detection of immunoblotted target protein bands, without hindrance by interfering immunoprecipitating immunoglobulin heavy and light chains. It is easy to generate publication-quality IP/Fluorescent Western Blot data with Mouse IgG TrueBlot®, simply substitute the conventional FITC Anti-mouse IgG blotting reagent with Fluorescent Mouse TrueBlot® Antibody Fluorescein and follow the prescribed protocol for sample preparation and immunoblotting. Mouse IgG TrueBlot® is ideal for use in protocols involving immunoblotting of immunoprecipitated proteins. TrueBlot preferentially detects the non-reduced form of mouse IgG over the reduced, SDS-denatured form of IgG. When the immunoprecipitate is fully reduced immediately prior to SDS-gel electrophoresis, reactivity of Mouse IgG TrueBlot® with the 55 kDa heavy chains and the 23 kDa light chains of the immunoprecipitating antibody is minimized thereby eliminating interference by the heavy and light chains of the immunoprecipitating antibody in IP/Western blot applications. Applications include studies examining post-translational modification (e.g., phosphorylation or acetylation) or protein-protein interactions.

Application Note

Fluorescent Mouse TrueBlot® Antibody Fluorescein has been tested in immunofluorescence, immunoprecipitation, and western blot. Fluorescein Conjugated Antibodies are designed for immunofluorescence microscopy, fluorescence based plate assays (FLISA), fluorescent western blotting, multiplex analysis, including multicolor imaging, utilizing various commercial platforms. Fluorescent Mouse TrueBlot® Antibody Fluorescein is provided as a lyophilized powder. To conserve reagent, we recommend incubating the blots from minigels in sealed bags (removing as much air as possible) with minimal volume (2-3 mLs). If used conservatively at 2.5mls/blot will yield enough reagent for 40 blots. Note that there are three key procedural considerations: 1. Protein A or G beads may be used with the mouse, goat and sheep TrueBlot secondaries, but not with the rabbit TrueBlot secondary. Use of protein A or G beads with the rabbit TrueBlot will result in contaminating bands. 2. Immunoprecipitate should be completely reduced. 3. MB-070 Blocking Buffer for Fluorescent Western Blotting should be used as the blocking protein for the immunoblot. Note: To achieve best results when detecting mouse IgG1 subtypes, we recommend performing a dot blot or titration to determine the optimal dilution factor for your desired application. All recommended dilutions for listed applications are intended as an initial recommendation, specific conditions for each protein and antibody combination should be specifically optimized by the end user.

Purity/Specificity

Fluorescent Mouse TrueBlot® Antibody Fluorescein Conjugate was prepared from tissue culture supernatant by Protein G affinity chromatography. Assay by Immunoelectrophoresis resulted in a single precipitin arc against anti-fluorescein and Anti-Mouse Serum. Reactivity is observed against native Mouse IgG by both Western blot and ELISA.